Millicell® devices using PET or CM membrane have been designed to allow visualization of cells from below using an inverted microscope. For viewing live cells, microscopic observations can be made through the receiver or plastic plate containing media. In order to focus on the cells, the microscope objective (typically 5–20X) must have an appropriate working distance. (For objective specifications, visit the websites listed in the Microscope Objective Information section.) Fixed cells that do not require to be visualized in media can be viewed directly without the receiver plate. However, care should be taken not to contaminate the objective with liquid residue (media, mounting fluid) on the membrane.
Some cell culture platforms can allow the cells to be viewed in a conventional microscope directly from above using low magnification. Cells can be visualized through the lid to maintain sterility or with the lid removed for fixed cells or when maintenance of sterility is not required. Working distances of the objective must be longer when reading from above compared to when reading from below. If using immunofluorescence, it is recommended to use a mounting fluid that contains an anti-fade additive to prevent photobleaching.
The membrane can be removed from each well for microscopic evaluation. This allows for higher magnification examination and storage of the slides for future use.
Figure 1. Visualizing Cells using the Millicell-24 Device
For visualizing from above the membrane, typically 5–20X objectives are used that have at least a 13.59 mm (A) or a 18.03 mm (B) working distance when viewing without or with the lid, respectively. For visualizing from below the membrane, 5–20X objectives are used that have at least a 2 mm (C) working distance.
Information regarding microscope objective magnification power and working distances can be obtained from individual optical dealers or from the microscope vendors:
NoteIt is assumed that users of this procedure will be knowledgeable in TEM procedures.
Figure 2. Living murine embryonic stem cell derived embryoid bodies visualized in a 1 um PET Millicell®-24 device using an Olympus IMT-2 inverted microscope
Figure 3. Neuron differentiation of embryonic stem cells in Millicell®-24 1 um PET filter plates.
Murine embryonic stem cells were formed into suspended embryoid bodies (EBs), then transferred to 1 um PET Millicell®-24 plates for attachment and differentiation. The photo inset shows the inverted phase contrast through membrane of live EBs in the media. Neural differentiation after netinoic acid treatment of attached EBs was confirmed by anti-neurofilament immunofluoresence.
Note: Steps 1–5 should be done on an intact Millicell®cell culture insert or plate well.
Note: It is not necessary to use any other agent, such as propylene oxide, with plastic. Propylene oxide will dissolve the cellulosic filters. In addition, the standard inversion/rotation of specimens used in these steps is not advised since either (1) damage to the cell layer or (2) stretching of the cellulosic filter may occur. Mild shaking on a gel shaker apparatus is sufficient for successful infiltration.
Note: Before the next step the membrane must be detached from the surrounding plastic ring. Sometimes this will occur without manipulation since the EPON may loosen the membrane-to-ring bond. If this does not occur, use a sharp scalpel or a cork borer and cut the membrane. It may also help to cut the membrane over a 47 mm filter support disk. Under no circumstances should the membrane be left attached to the ring during polymerization.
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